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Cite this: NewJ.Chem., 2016,

40, 5885

Coordination compounds of a hydrazonederivative with Co(III), Ni(II), Cu(II) and Zn(II):synthesis, characterization, reactivity assessmentand biological evaluation†

B. Barta Hollo,a J. Magyari,a S. Armakovic,b G. A. Bogdanovic,c M. V. Rodic,a

S. J. Armakovic,a J. Molnar,d G. Spengler,d V. M. Leovaca andK. Meszaros Szecsenyi*a

A new hydrazone-type ligand with a five N-donor set and its coordination compounds with Co(III), Ni(II),

Cu(II) and Zn(II) metal centres were synthesized. The crystal and molecular structure of the Co(III) complex

was determined by X-ray structural analysis. All the compounds were characterized by elemental analysis,

molar conductivity and FT-IR spectral data. The cobalt(III) compound is a neutral binuclear complex

formed by coordination of two, doubly deprotonated ligand anions as bridges between the Co(III) centres.

The metal centres are additionally connected by a peroxido bridge, which was observed for the first time

in Co(III) complexes with similar ligands. The other coordination compounds are mononuclear complexes

wherein only one doubly deprotonated ligand is coordinated to the central ion in a tetradentate fashion.

The structures were theoretically investigated employing density functional theory (DFT) calculations with

B3LYP exchange–correlation functional and LACV3P+(d,p) basis sets for the coordination compounds

and 6-31+G(d,p) basis set for the ligand. Molecular electrostatic potential (MEP) and average local

ionization energy (ALIE) surfaces were calculated to study the reactive properties of the compounds. The

thermal behaviour of the compounds was determined by simultaneous thermogravimetric-differential

scanning calorimetric (TG-DSC) measurements and the results were correlated to the proposed

structures and to calculated MEP/ALIE surfaces. The compounds were tested in vitro for antiproliferative

effects on parental (L5178Y) mouse T-cell lymphoma cells, on L5178Y transfected with pHa ABCB1/A

retrovirus and for reversal of multidrug resistance (MDR) in tumor cells by flow cytometry. The

preliminary measurements showed that the cobalt(III) compound was an effective inhibitor of the ABC

transporter PGP drug efflux pump. The ligand was somewhat less effective, the zinc complex had a

moderate inhibition activity, whereas the nickel(II) and copper(II) compounds were inactive.

Introduction

Cancer mortality accounts for about one eighth of all deaths inthe world. During the past 30 years, the annual cancer incidence

has doubled. Around 1.5 million new cancer cases and 0.6 millioncancer deaths are projected to occur in the United States in 2015.1

Carcinogenesis underlies complex mechanisms and to addresssingle target approaches is inadequate to prevent prevalence anddeaths from the disease. Adjuvant therapy of cancer may decreasethe development of drug resistance to some extent. However,multidrug resistance (MDR) caused by specific membranetransporters, such as ATP-binding cassette (ABC) or coppertransporters, as well as other causes of drug resistance, hampersuccessful cancer chemotherapy.2

Numerous pyridazine derivatives are biologically active. Inaddition to the natural products with pyridazine, a high numberof synthetic derivatives are in use as pharmaceuticals for treatmentof inflammations, pain, enzyme dysfunctions and tumors.3a–d Formore than ten years, Dal Piaz et al. studied the pyridazine derivativeswith antinociceptive activity.4a,b Pyridazine-type vasodilators are

a University of Novi Sad, Faculty of Sciences, Department of Chemistry,

Biochemistry and Environmental Protection, Trg Dositeja Obradovica 3, 21000,

Novi Sad, Serbia. E-mail: mszk@uns.ac.rsb University of Novi Sad, Faculty of Sciences, Department of Physics,

Trg Dositeja Obradovica 4, 21000, Novi Sad, Serbiac ‘‘Vinca’’ Institute of Nuclear Sciences, Laboratory of Theoretical Physics and

Condensed Matter Physics, University of Belgrade, P.O. Box 522, 11001 Belgrade,

Serbiad University of Szeged, Faculty of Medicine, Department of Medical Microbiology

and Immunobiology, Dom ter 10, H-6720 Szeged, Szeged, Hungary

† Electronic supplementary information (ESI) available. CCDC 1432904 (1). For ESI andcrystallographic data in CIF or other electronic format see DOI: 10.1039/c6nj00560h

Received (in Victoria, Australia)19th February 2016,Accepted 26th April 2016

DOI: 10.1039/c6nj00560h

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also described.5a,b The relatively simple structure and highreactivity of pyridazines make them good candidates for synthesisof more complex heterocyclic systems. The coordination chemistryof pyridazine-based compounds is sparsely covered and mainlyfocused towards selected heavy metals6a–e although they aresuitable for coordination complex formation.7a,b The activity ofbiologically active compounds can be modified by coordinationto transition metal ions.8a–c Sometimes the activity of the ligandis increased by its coordination,9a,b but the opposite effect isalso observed.10 Schiff base-type ligands and their coordinationcompounds display a wide spectrum of biological activity rangingfrom antimicrobial effect11 and cytotoxicity against various cancercells.12a,b

The reaction between 3-chloro-6-hydrazinopyridazine (Hp) with2,6-diacetylpyridine results in the formation of a new potentiallypentadentate Schiff base-type ligand bis(3-chloropyridazine-6-hydrazon)-2,6-diacetylpyridine (Hp2DAP). We present herein thesynthesis, structural and physicochemical properties of the Hp2DAPligand and its complexes with Co(III), Ni(II), Cu(II) and Zn(II) metalcentres, [Co2(m-Hp2DAP-2H)2(m-O2)]�4H2O (1), [Ni(Hp2DAP-2H)] (2),[Cu(Hp2DAP-2H)]�H2O (3) and [Zn(Hp2DAP-2H)]�H2O (4),respectively. Density functional theory (DFT) calculations wereperformed on all the compounds and the molecular electro-static surfaces (MEP) and average local ionization energy (ALIE)surfaces were calculated to determine the reactivity centres.The compounds were tested with resistance modifier steroidcompounds to overcome drug resistance in resistant humanABCB1-expressing mouse lymphoma cells. The structural dataand the calculated MEP and ALIE surfaces were correlated tounderstand the thermal properties and the biological activity ofthe compounds.

ExperimentalSynthesis of the compounds

Hp2DAP. After dissolving 5 mmol (0.726 g) of 3-chloro-6-hydrazinopyridazine (Hp) in 10 cm3 acetonitrile under reflux at60 1C, 2.5 mmol (0.410 g) of 2,6-diacetylpyridine (DAP) dissolved in5 cm3 acetonitrile was added. The reaction mixture was diluted by2 cm3 acetonitrile and the reflux continued for 30 min at 60 1C. Alight orange amorphous precipitate was filtered after 24 h, washedwith acetonitrile and dried in air at room temperature. Yield:93.6%, Mr = 416.27 (found: C, 49.1; H, 3.6; N, 30.3, calc. forC17H15Cl2N9 C, 49.10; H, 3.82; N, 30.00%). lM (S cm2 mol�1) = 0.74.

The coordination compounds were prepared by the followinggeneral procedure:

The ligand, Hp2DAP (0.25 mmol, 0.104 g) was suspended in15 cm3 methanol by heating and intensive stirring. To the warmsuspension, a warm solution of Co(II), Ni(II) or Cu(II) acetate(0.25 mmol) in 5 cm3 methanol was added. The reactionmixture was heated and stirred until the dissolution of theligand. After about 4 days, the crystals formed were filtered-off,washed with methanol and dried in air at room temperature.

The compound with Zn(II) was prepared similarly, exceptthat instead of zinc acetate, zinc chloride was used. When the

ligand fully dissolved and the color changed to dark red thereaction mixture was cooled to room temperature. To the coolsolution, to adjust the pH, 3 cm3 of NH3(aq) was added. A darkred microcrystalline product started to precipitate and wascompleted in 24 hours.

Analytical data

[Co2(l-Hp2DAP-2H)2(l-O2)]�4H2O (1). Color: dark red(almost black). Yield: 32.3%. Mr = 1050.44 (found C, 38.9; H,3.3; N, 24.0, calc. for C34H26Cl4Co2N18O2�4H2O C, 38.45; H,3.20; N, 23.66%). lM (S cm2 mol�1) = 0.50.

[Ni(Hp2DAP-2H)] (2). Color: dark brown microcrystallineprecipitate. Yield: 80.0%. Mr = 474.97 (found C, 43.0; H, 3.2;N, 26.5, calc. for C17H15Cl2N9Ni C, 42.00; H, 2.89; N, 25.63%).lM (S cm2 mol�1) = 0.20.

[Cu(Hp2DAP-2H)]�H2O (3). The reaction mixture was dilutedby 5 cm3 methanol. Color: dark brownish-red microcrystallineprecipitate. Yield: 71.5%. Mr = 497.83 (found C, 41.0; H, 3.4; N,25.3, calc. for C17H15Cl2CuN9.H2O C, 41.78; H, 3.10; N, 25.07%).lM (S cm2 mol�1) = 0.00.

[Zn(Hp2DAP-2H)]�H2O (4). Color: dark red microcrystallineprecipitate. Yield: 75.2%. Mr = 499.68 (found C, 40.9; H, 3.4; N,25.2 calc. for C17H15Cl2N9Zn�H2O C, 41.26; H, 3.11; N, 25.35%).lM (S cm2 mol�1) = 0.20.

Physical measurements

IR data were collected on a Thermo Nicolet Nexus 670 FT-IRspectrometer at room temperature in the range of 4000–400 cm�1

with resolution of 4 cm�1 using KBr pellets.Raman spectra were obtained with the LabRAM system

(Horiba Jobin-Yvon, Lyon, France) coupled with an external532 nm Nd-YAG laser source (Sacher Lasertechnik, Marburg,Germany) and an Olympus BX-40 optical microscope (Olympus,Hamburg, Germany). The resolution of the Raman instrumentwas ca. 3 cm�1 in the 4000–200 cm�1 spectral range and abackscattered geometry was used. For the measurements solidsamples were used.

The molar conductivity of freshly prepared 1 � 10�3 mol dm�3

solutions of the complexes in N,N0-dimethylformamide (DMF) wasdetermined at room temperature using a digital conductivity meter(Jenway 4010).

TG/DSC measurements were carried out on a TA Instru-ments SDT Q600 thermal analyzer with 20 1C min�1 heatingrate in the range from room temperature to 700 1C and samplemass o3 mg. Carrier gases: nitrogen, air. Gas flow rate:100 cm3 min�1. Sample holder/reference: alumina crucible/empty alumina crucible.

Cell lines and cultures

L5178Y parental (PAR) mouse T-cell lymphoma cells (ECACCcat. no. 87111908; U.S. FDA, Silver Spring, MD, USA) weretransfected with pHa MDR1/A retrovirus as described previously.2

The ABCB1-expressing cell line (MDR) was selected by culturingthe transfected cells with 60 ng cm�3 of colchicine (Sigma-AldrichChemie GmbH, Steinheim, Germany) to maintain the MDRphenotype. The cells were cultured in McCoy’s 5A medium (Sigma)

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supplemented with L-glutamine (Sigma) and antibiotics(penicillin/streptomycin solution, Sigma) at 37 1C and in anatmosphere with 5% CO2.

Assay for antiproliferative and cytotoxic effect

The effects of increasing concentrations of the compounds oncell growth were tested in 96-well flat-bottomed microtiterplates. The compounds were diluted in 100 mL medium then6 � 103 cells for antiproliferative assay or 2 � 104 cells forcytotoxic assay in 100 mL of medium, were added to each well,with the exception of the medium control wells. The compoundswere dissolved in DMSO. The solvent applied in the assays had noeffect on cell growth. The culture plates were further incubated at37 1C for 72 h and 24 h. At the end of the incubation period 20 mLof 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide(MTT, Sigma-Aldrich Chemie GmbH) solution (from a 5 mg cm�3

stock) were added to each well. After incubation at 37 1C for 4 h,0.10 cm3 of sodium dodecyl sulfate (SDS, Sigma) solution (10%in 0.01 mol dm�3 HCI) were added to each well and the platesfurther incubated at 37 1C overnight. The cell growth was thendetermined by measuring the optical density (OD) at 550 nm(ref. 630 nm) with a Multiscan EX ELISA reader (ThermoLabsystems, Cheshire, WA, USA).

Assay for reversal of MDR in tumor cells by flow cytometry

L5178Y parental (PAR) and multidrug resistant (MDR) mouse T-celllymphoma cells were adjusted to a density of 2 � 106 cells cm�3,resuspended in serum-free McCoy’s 5A media and distributed in0.5 cm3 aliquots (1 � 106 cells). The cells were incubated in thepresence of the ligand and its complexes for 10 min at roomtemperature. Then, the fluorescent ABCB1 substrate rhodamine123 (R123, Sigma-Aldrich) was added to each sample at a finalconcentration of 5.2 mM and the cells were incubated for 20 minat 37 1C in a water bath, washed twice and resuspended in0.5 cm3 phosphate-buffered saline (PBS) for analysis. Thefluorescence of the cell population was measured with a PartecCyFlow flow cytometer (Partec, Munster, Germany). Verapamilhydrochloride (EGIS Pharmaceuticals PLC, Budapest, Hungary)was used as a positive control in MDR cells at a final concen-tration of 10 mg cm�3.13

Representative flow cytometric experiments with 1 � 104 cellswere evaluated for the accumulation of R123.13 The fluorescenceactivity ratio (FAR) for L5178Y/PAR and L5178Y/MDR cell lines wascalculated using the following equation:

FAR ¼ MDRtreated=MDRcontrol

Parentaltreated=Parentalcontrol

The results provided are from a representative flow cytometricexperiment in which 10 000 individual cells were evaluated.Partec CyFlow flow cytometer research software was used toanalyze the recorded data. The data were first presented ashistograms and were converted to FAR units that define fluorescenceintensity, standard deviation, and peak channel in the total and inthe gated populations.

X-ray crystallography

Single-crystal X-ray diffraction data for 1 were collected at roomtemperature on an Oxford Diffraction Gemini S diffractometerwith Mo-Ka radiation (l = 0.71073 Å). Data collection, unitcell finding, intensity integration and scaling of the reflectionswere performed with the CRYSALISPRO software.14 Empiricalabsorption correction using spherical harmonics, implementedin the SCALE3 ABSPACK scaling algorithm within CRYSALISPROwas applied. The crystal structure was solved with SHELXT15

and refined using SHELXL-201416 with SHELXLE17 as thegraphical interface.

All non-hydrogen atoms were refined anisotropically. Thehydrogen atoms attached to the carbon atoms were placed atgeometrically idealized positions with C–H distances fixed to0.93 and 0.96 Å and their isotropic displacement parameters setequal to 1.2 Ueq and 1.5 Ueq of the parent sp2 and sp3 C atoms,respectively. X-ray diffraction data did not allow unambiguousdetermination of the H-atoms bonded to crystal water moleculesin difference electron density maps.

A summary of the pertinent crystallographic data are givenin Table 1. The ORTEP-3 and WINGX software were used for thepreparation of the materials for publication.18

Computation details

All density functional theory (DFT) calculations were performedusing the Jaguar 8.7 program,19 as implemented in the SchrodingerMaterial Suite release 2015-1 (Schrodinger, LLC, New York, NY,2015). DFT calculations for structures containing transition metalswere performed using B3LYP20a–c exchange–correlation functional

Table 1 Crystallographic data for crystal structure of 1

Molecular formula C34H26Cl4Co2N18O2�4H2OFormula weight 1050.45Color, crystal shape Dark red, prismCrystal size (mm3) 0.52 � 0.13 � 0.04Temperature (K) 293(2)Wavelength (Å) 0.71073Crystal system MonoclinicSpace group C2/ca (Å) 12.7108(4)b (Å) 22.8673(5)c (Å) 14.7326(3)a (1) 90b (1) 94.369(2)g (1) 90V (Å3) 4269.76(19)Z 4Dcalc (Mg m�3) 1.634m (mm�1) 1.095y range for data collection (1) 3.12–29.07Reflections collected 16 829Independent reflections, Rint 5072, 0.0275Completeness to y = 26.321 (%) 99.9Data/restraints/parameters 5072/0/291Goodness-of-fit on F2 1.053Final R1, wR2 indices (I 42sI) 0.0375, 0.0934Final R1, wR2 indices (all data) 0.0548, 0.1007Largest diff. peak & hole (e Å�3) 0.402 and �0.241

where R1 =P

||Fo| � |Fc||/P

|Fo|; wR2 = [P

(w(Fo2 � Fc

2)2)/P

(wFo2)2]1/2;

GoF = [P

(w(Fo2 � Fc

2)2)/(n � p)]1/2, n is the number of reflections, andp is the number of refined parameters.

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with LACV3P+(d,p) basis set, whereas the ligand structure wasinvestigated again using the B3LYP exchange–correlation functionalwith 6-31+G(d,p) basis set. A C2 symmetry constraint duringgeometrical optimization was employed for the binuclearcobalt(III) complex 1, whereas the other assumed structureswere geometrically optimized without symmetry constraints.After successful geometrical optimization, a single point energycalculation was run to obtain additional information on molecularelectrostatic potential (MEP) and average local ionization energy(ALIE) surfaces.

Results and discussionSynthesis of compounds

The bis(3-chloropyridazine-6-hydrazone)-2,6-diacetylpyridine(Hp2DAP) ligand was synthesized according to the proceduredescribed in our previous study10 but instead of methanol thereactions were carried out in acetonitrile. In the reactionbetween the hydrazine group of 3-chloro-6-hydrazinopyridazineand the carbonyl groups of 2,6-diacetylpyridine a new Schiff-basewas formed with five potential coordinating N atoms (Fig. 1).

Hp2DAP is not soluble in methanol, but its warm suspensionreacts with transition metal ions in the presence of a protonacceptor. Therefore, for the synthesis, the acetate salts of Co(II),Ni(II) and Cu(II) were used. In the case of Zn(II), the salt waszinc(II) chloride and the deprotonation of Hp2DAP to Hp2DAP-2H2�

was promoted by the addition of ammonia.21 With Ni(II), Cu(II) andZn(II), the expected neutral coordination compounds were formedbut with cobalt(II) acetate the reaction route was different. In air,during the complex formation, Co(II) was oxidized to Co(III)and water to peroxide and a dimeric coordination compound[Co2(m-Hp2DAP-2H)2(m-O2)]�4H2O (1) with a peroxido bridgebetween the Co(III) centres was formed. The reaction is highlyrepeatable and probably occurs by a complex mechanism, yet theyield is rather low, compared to the yields of the other com-pounds. The molar conductivity data refer to the non-electrolyticcharacter of all four coordination complexes, in accordance withthe coordination of the ligand in its doubly deprotonated form.22

Crystal and molecular structure of complex 1

Single-crystal X-ray analysis shows that the complex of Co(III)and Hp2DAP ligand (1) is a neutral binuclear coordinationcomplex with two symmetrically related halves (Fig. 2 and 3).

The crystallographic asymmetric unit also includes two watermolecules of crystallization and therefore the crystal structurecan be described by the formula [Co2(m-Hp2DAP-2H)2(m-O2)]�4H2O. Hp2DAP contains two arms bonded to the centralpyridine ring in the same positions regarding the N1 nitrogenatom. These two arms are denoted as A and B in Fig. 2 and 3using the suffixes a and b in atomic labels. A and B have equalcompositions but very different orientations with regard to thepyridine ring. While arm A is approximately coplanar with thepyridine ring, arm B is considerably displaced from the plane ofthe central ring (Fig. 2). This structural difference between Aand B can be illustrated well by comparison of the N1–C–C6–N2torsion angles [�1.4(3)1 and 71.1(3)1 for A and B, respectively]

Fig. 1 Structure of Hp2DAP.

Fig. 2 Asymmetric unit of complex 1 with atom numbering scheme.Crystal water molecules are omitted for clarity. The Co1i and O1i atoms[symmetry code: (i) �x, y, �z + 0.5] do not belong to the same asymmetricunit. Displacement ellipsoids of non-H atoms are drawn at the 40%probability level.

Fig. 3 Binuclear unit of complex 1. H atoms and water moleculesare omitted for clarity. Displacement ellipsoids are drawn at the 30%probability level.

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and dihedral angles between pyridine ring and the terminalN4–N5 ring [10.36(15)1 for A and 79.29(9)1 for B]. All non-Hatoms are roughly coplanar in both moieties but the fragmentB is somewhat more puckered as is mainly demonstrated inthe value of the C6–N2–N3–C8 torsion angles [179.1(2)1 and�164.5(2)1 for A and B, respectively].

All the abovementioned structural differences between armsA and B can be explained by their different coordinationbehaviour. Namely, the A moiety is coordinated to the Co atomtogether with the N1 atom, whereas the B fragment is coordinatedto the neighbouring Co1i atom [symmetry code: (i) �x, y, �z + 0.5]forming a bridge between the two metal atoms in the binuclearunit (Fig. 2 and 3). The moiety A together with the pyridinenitrogen form two fused five-membered chelate rings that arealmost ideally coplanar [dihedral angle between two chelaterings is 2.11(13)1]. The B fragment is coordinated to the metalatom through the same nitrogen atoms, the N2 and N4, as is thecase for A. However, the Co–N2 coordination bond for B isalmost 0.13 Å longer than the corresponding bond for the Amoiety. Such a high difference between the two equal coordinationbonds is also surprising because the N2a and N2b atoms arechemically identical and (mutually) situated in a trans positionregarding the Co1 atom. An explanation could be found in thesupposed orientation of the nitrogen’s (N2a) lone electronpair regarding the metal atom. Because the N2 atom is sp2

hybridized it is reasonable to expect that its lone pair lies in thesame plane as the C6–N2–N3 fragment. In the case of the Afragment, the Co atom lies perfectly in the C6–N2–N3 plane andconsequently the location of the metal atom corresponds verywell with the orientation of the nitrogen’s lone pair. Thisenables a relatively strong Co–N2a coordination bond. In thecase of fragment B, the Co atom is significantly displaced fromthe C6–N2–N3 plane [displacement of 0.205(7) Å] and consequentlythe Co1–N2b bond is significantly weaker and longer thanthat in fragment A [1.8691(18) Å for A and 1.9945(17) Å for B].Herein, we can also add the information that the Co–N2aand corresponding Co–N2b are the shortest and the longestcoordination bonds, respectively, in the crystal structure ofcomplex 1 (Table 2).

The interesting coordination mode of the Hp2DAP ligand inthe present structure and the different structural behaviourof the two moieties are directly related to the coordinationgeometry of the Co(III) atom. The metal atom in 1 is coordinatedin a rather distorted octahedral arrangement by the N5O donorset (Fig. 4). The N2a–Co1–N4a, N1–Co1–N2a and N2b–Co–N4bcoordination angles (Table 2) significantly deviate from 901,whereas the N1–Co1–N4a coordination angle of 1621 considerablydeviates from an ideal value of 1801. This could be expected due toformation of five-membered chelate rings (influence of two fusedchelate rings in the case of the N1–Co1–N4a angle). However, somecoordination planes are also significantly deformed. Thus, theCo1, N1, N2a, N4a are nearly coplanar (root-mean-square deviationof fitted atoms is only 0.012 Å), whereas the N2bi atom, whichlies in the same coordination plane, is displaced from theCo1–N1a–N2a–N4a mean plane by 0.286(3) Å. The two equivalentangles, C1–N1–Co1 and C5–N1–Co1, should have very similar

values but in the crystal structure of 1 these bond angles arevery different [130.19(16)1 and 111.15(15)1, respectively]. Thismeans that the Co1–N1 coordination bond does not coincidewell with the direction of the lone pair of the N1 atom.

The abovementioned analysis demonstrates very interestingstructural characteristics and exceptional flexibility of theHp2DAP ligand. In the Cambridge Structural Database (CSD,Version 5.34, May 2013),23 we found 150 crystal structures oftransition metal complexes with very similar ligands to complex1 (Fig. 4a). The Hp2DAP derivatives are mainly coordinated aspentadentate ligands (116 crystal structures) with the coordinationmode illustrated in Fig. 4b. Binuclear complexes are not rare butcomplex 1 is the only example, which contains a peroxido bridgebetween two metal centres (O1–O1i in Fig. 3). In the CSD, thereare 26 crystal structures that contain the fragment shownin Fig. 4a and the Co metal atom. All Co(II) complexes havecoordination mode, as shown in Fig. 4b, and only one of them is

Table 2 Selected bond distances (Å) and coordination angles (1) in thecrystal structure of complex 1. Symmetry code: (i) �x, y, �z + 0.5

Bond length Å Bond angle 1

Co1–N2a 1.8691(18) N2a–Co1–N4a 80.52(8)Co1–O1 1.8843(14) O1–Co1–N4a 88.49(7)Co1–N4a 1.9187(18) N2a–Co1–N4bi 93.04(8)Co1–N4bi 1.9399(19) O1–Co1–N4bi 176.30(7)Co1–N1 1.9685(18) N4a–Co1–N4bi 94.73(8)Co1–N2bi 1.9945(17) N2a–Co1–N1 82.17(8)O1–O1i 1.416(3) O1–Co1–N1 89.66(7)N1–C1 1.339(3) N4a–Co1–N1 162.62(8)N1–C5 1.369(3) N4bi–Co1–N1 87.81(8)N2a–C6 1.290(3) N2a–Co1–N2bi 170.29(8)N2a–N3a 1.357(3) O1–Co1–N2bi 97.88(7)N3a–C8a 1.331(3) N4a–Co1–N2bi 93.06(7)N4a–N5a 1.343(3) N4bi–Co1–N2bi 80.16(8)N4a–C8a 1.362(3) N1–Co1–N2bi 104.32(7)N2b–C6b 1.296(3)N2b–N3b 1.391(3)N3b–C8b 1.336(3)N4b–C8b 1.350(3)N4b–N5b 1.350(3)C1–C6b 1.479(3)C5–C6a 1.446(4)

Fig. 4 (a) Structural fragment used in the CSD search. (M is any transitionmetal, X is an N, O or S atom). (b) The most frequent coordination modefound in 116 crystal structures, which contain the fragment is shown in (a).All donor atoms are roughly coplanar with the metal atom.

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binuclear24 but with the same coordination mode. There areadditional two binuclear complexes,23,25 both with Co(III) metalcentres and have, as well as Co(II) binuclear complexes, differentcoordination modes than that presented in 1. Recently, we havereported the crystal structure of a Co(III) complex10 with thesame donor set and very similar ligand to Hp2DAP as in 1, but inthat case the complex molecule crystallized as discrete mono-nuclear units. A comparison of structural data for all 150structures extracted from CSD also showed that complex 1,along with some Ni complexes26a–d form one of the shortestM–N2 bonds in transition metal complexes with DAP deriva-tives. One can conclude that the presented binuclear Co(III)complex 1 has some interesting structural characteristics anda coordination mode, which is observed for the first time in Cocomplexes with similar ligands.

IR spectral data

In the ligand, the characteristic nNH bands appear at ca.3400 cm�1. Due to the deprotonation of the two N atoms of thetwo pyridazine rings Hp2DAP coordinates through deprotonated Natoms. The intensity of the skeleton vibration bands in the regionsof B1400–1500 cm�1 and B1100–1200 cm�1 increases and in thespectra of 2, 3 and 4 the bands are overlapped compared to thosein the ligand. The characteristic vibrations for the compounds arelisted in Table 3.

As a consequence of complex formation, the change in themutual orientations of the methyl groups results in additionalnCH vibrations appearing in the spectra of the complexes,especially in that of 1. In the spectrum of 1, further bandsplitting is observed related to its dimeric structure. Thesedifferences prove the coordination of the ligand through itsN atoms and refer to the structural differences between 1 andthe other three coordination complexes. The characteristicvibration around 860 cm�1 in 1 for the –O–O– group is activein the Raman due to its symmetry. The ligand and coordinationcomplexes 1 and 4 were also characterized by Raman spectra(Fig. 5). The other two compounds 2 and 3 were not suitable forRaman measurements. For the detection of the peroxido bridge,the most interesting region is in the range from B900 cm�1 toB400 cm�1. The Raman band of m-O2 appears at lower energythan the band of the side-on peroxido groups,27 which usuallyappears somewhat above 850 cm�1.28a,b In the spectrum of 1(Fig. 5), the most intensive sharp peak at 691 cm�1 and thenext one at 621 cm�1 can be assigned as peroxide vibrations,whereas the other two peaks presumably belong to Co–Ovibrations. Comparing all three spectra, it is obvious that

compound 4 gives similar bands as the ligand and does notcontain a peroxido group.

Results of DFT calculations: MEP and ALIE surfaces

To obtain an insight into the reactive properties of the structurescomputationally investigated in this study we shall refer to theMEP and ALIE surfaces.

MEP surfaces present a very useful descriptor to determinepotential sites prone to electrophilic attack and nucleophilicreactions.29a–c This descriptor is important as it is related to thecharge distribution of the investigated molecule. EmployingMEP, one is able to determine how molecules interact withother molecules or radicals. The MEP (hereafter indicated asV (r)) is given as follows:

Vð~r Þ ¼X ZA

RA �~rj j �ð

r ~r 0ð Þ~r 0 �~rj j d~r (1)

where the summation runs over all nuclei A with charge ZA andcoordinate RA, whereas r(r0) is the electron density of themolecule. V(r) represents the potential exerted at the coordinater by the nuclei and the electrons. The sign of V(r) at any pointdepends on whether the effects of the nuclei or the electronsare dominant.30a,b The last equation is valid when polarizationand nuclear rearrangement effects due to the presence of a unittest charge at the distance r are neglected.

Another very useful indicator of molecule’s reactivity is itsALIE surface. ALIE is defined as a sum of orbital energies

Table 3 Characteristic FTIR bands of the compounds

Vibration Hp2DAP [cm�1] 1 [cm�1] 2 [cm�1] 3 [cm�1] 4 [cm�1]

nC–H 3430–2850 3410–2840 3430–2850 3435–2850 3425–2845nring 1590–1500 1590–1500 1580–1495 1580–1495 1585–1495nCQN 1450–1400 1465–1395 1465–1385 1460–1390 1450–1395nCAr–N 1370 1335–1285 1325–1285 1330–1265 1325–1285dring, dCQN, nCQN, dCH3 1130–1020 1160–1040 1165–1025 1145–1025 1150–1025tN–H 875–805 — — — —nC–Cl 730–675 750–645 730–635 730–635 730–630

Fig. 5 The Raman spectra of Hp2DAP, 1 and 4.

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weighted by the orbital densities31a,b and this approach representsan energetic measure of how easy or difficult is it to removeelectrons from certain areas of the molecule. It was demonstratedby Sjoberg31 and Murray32 that the ALIE I(r) plotted on a molecularsurface is a fine strategy to understand chemical reactivity inaromatic systems.29b,32 I(r) is defined in the following manner:

IðrÞ ¼Xi

rið~r Þ eij jrð~r Þ (2)

where ri(-r ) is the electronic density of the i-th molecular orbital

at the point -r, ei is the orbital energy, while the r(-r ) is the total

electronic density function. Using Koopman’s theorem, I(r) canbe interpreted as the average energy needed to ionize anelectron at any particular point -

r in the space of a molecule.Therefore, the locations in the molecule wherein the ALIE has thelowest values represent areas of the least tightly bound electrons andsites to be most reactive towards electrophiles.29b,30b The equilibriumgeometries and the MEP and ALIE surfaces for 1 and Hp2DAP arepresented in Fig. 6. It should be noted that both MEP and ALIEsurfaces were mapped on the electron density surface.

The MEP surface of Hp2DAP indicates that the lowestelectrostatic potential is located (to small extent) in the nearvicinity of the central nitrogen (N2) atom. However, the ALIE surfaceof the ligand identifies the locations of nitrogen atoms as centressuitable for electrophilic attack. Electron density marked by a redcolor is located in a significant amount precisely on these atoms.

In 1 both MEP and ALIE surfaces indicate that from theaspect of electrophilic attacks, the important reaction sitescould be the central part of complex at the location whereinthe oxygen atoms are placed. According to the MEP surface, theoxygen atoms should be considered as the most importantreaction sites, whereas the ALIE surface indicates that nitrogenatoms also represent potential reaction sites, because at theselocations, the electrons are also less tightly bound.

The proposed geometries for compounds 2–4 are depicted inFig. 7. The lowest values of MEP for the proposed structures arepractically the same for the coordination compounds 2–4. Onthe other hand, the maximum value of MEP for the structurecontaining the Zn(II) atom (4, Fig. 7) is two times higher thanthat for 2 and 3 (Fig. 7), whereas the latter two have almostidentical values for maximum values of the MEP. Concerningthe charge distribution according to the MEP surfaces, it can beobserved that in all cases, the nitrogen atoms of the ligand,which are not coordinated are recognized as significant reactionsites, as at these locations MEP has the lowest values (indicatedby the red color in Fig. 7). The lowest values of MEP can befound in the pocket around the central atoms in 2–4. Thehighest value of MEP among all three mononuclear complexesis found in 4 and is located practically on the zinc atom (Fig. 7).

ALIE surfaces also indicate the reactive significance of nitrogenatoms and the neighbouring metal centres, as at these locationsthe electrons are the least tightly bound (indicated by the red colorin Fig. 7), which is in agreement with the MEP surfaces. In the caseof 2, significant red color is located precisely at the nickel atom.Moreover, the yellow-to-red color of ALIE surfaces located in the

near vicinity of other nitrogen atoms for the calculated structures2–4 identifies these sites as interesting ones from the aspect ofelectrophilic attacks.

In Table 4, the calculated dipole moment, m, the polar surfacearea (PSA) and the total surface area (TSA) are presented along withthe molecular lipophilicity.33 The measure of the molecular lipo-philicity is the log P (the logarithm of the 1-octanol/water partitioncoefficient) marked as A log P.

The influence of DMSO solvent on the geometries of thecomplexes has also been investigated through DFT calculations(for the details, see ESI†). It was found that although the solventsignificantly rotated the pyridazine moiety around the N–Nbond, complexes 2 and 3 preserved their square-planar coordinationgeometry. For 4, geometrical optimizations with the solvent resultedin unreasonable geometries. This phenomenon is most probablydue to the proposed unfavorable square-planar geometry aroundthe zinc centre.

Thermal data for Hp2DAP and 1–4

The thermal stability of new compounds is sometimes crucialfor their potential application.34 In addition, thermal data may

Fig. 6 Geometries of optimized Hp2DAP and 1 and the correspondingMEP and ALIE surfaces.

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be valuable in detecting even small structural differences.35 Asonly the molecular structure of 1 was determined by X-ray

structure analysis, the compounds were characterized also bymeans of TG-DSC measurements and their thermal decompositionpatterns were compared.

The Hp2DAP ligand is stable up to 200 1C and its thermaldecomposition does not depend on the atmosphere up to400 1C. A sharp endothermic effect around 300 1C refers tothe decomposition accompanied by melting of the ligand,which was also observed visually.

The heat effects accompanying the decomposition reactionsin complex compounds in a nitrogen atmosphere are presentedby the corresponding DSC curves in Fig. 8. The structural

Fig. 7 Geometries of the proposed structures for 2, 3 and 4 and the corresponding MEP and ALIE surfaces.

Table 4 Calculated dipole moment, polar surface area, total surface areaand A log P of Hp2DAP and 1–4

Sample code m [D] PSA [Å2] TSA [Å2] A log P

Hp2DAP 2.23 119.69 664.73 3.221 0.24 147.10 924.91 3.702 8.56 76.00 617.12 0.333 8.56 76.00 631.31 0.334 8.84 76.00 642.65 0.33

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preferences of the central atoms can be clearly observed in thecorresponding DSC curves. In compound 1, with a dimericstructure and a peroxido bridge, the most favorable octahedralsurround is established around cobalt(III). Moreover, bothdeprotonated Hp2DAP-2H2� are coordinated to both Co(III)centres as bridges. This arrangement results in relatively lowrepulsions between the ligands. The peroxido group is heatsensitive; therefore, the decomposition of anhydrous 1 startswith exothermic processes due to oxidation reactions with theperoxido group. These reactions are missing in compounds 2–4(Fig. 8). The strong electrostatic interactions (supported by thehigh value of MEP around the Zn metal centre) result in thesignificantly higher thermal stability of 4. A similar effect wasobserved in the case of a Zn(II) complex with bis(phthalazine-1-hydrazone)-2,6-diacetylpyridine.10

Cytotoxic activity evaluation

The biological activity of Hp2DAP and its coordination compoundswith Co(III) 1, Ni(II) 2, Cu(II) 3 and Zn(II) 4 was studied onantiproliferative effects, cytotoxic effects and inhibitory effectof the ABC transporter P-glycoprotein encoded by human MDR1gene on tumor cells. This transporter protein is responsible forthe pumping out, i.e. the removal of anticancer drugs from thetumor cells and as a consequence the cell will overgrow despitethe chemotherapy. For these in vitro experiments, the humanMDR1 gene transfected mouse lymphoma cells were applied. Inthe flow cytometry, the effects of the compounds were studiedon the drug accumulation in MDR cells in short (20 minutes)experiments in flow cytometry. The rhodamine 123 accumula-tion in MDR1 gene transfected cells was reduced in the presenceof Hp2DAP, 1 and, to some extent, 4; however, complexes 2 and 3were ineffective in the applied 8 mg cm�3 concentration when theresults were compared to the positive control, Verapamil, appliedin a concentration of 10 mg cm�3. The results are shown inTable 5.

Apparently, there is some structure–activity relationship betweenthe chemical structures of the hydrazine derivative and itscoordination complexes and the inhibition of PGP-170(P-glycoprotein, PGP, ABCB1, MDR1, Mr B 170) mediated multi-drug resistance of cancer cells. The cytotoxic effects of all thecompounds measured on the parent drug sensitive PAR anddrug resistant MDR cells showed no difference between the

sensitivity of the two cancer cell lines, meaning they are notsubstrates for the MDR1 gene-coded drug efflux.

Significant differences were detected for the compoundsregarding the antiproliferative effects and the drug accumulationin MDR cells. The evidence shows that the cobalt(III) compound 1was the most effective inhibitor of the ABC transporter PGP drugefflux pump that is responsible for extruding the anticancer drugsfrom cancer cells in in vitro studies. The zinc(II) complex 4 showedabout half the effect when compared to 1, whereas the compoundswith Ni(II) 2 and Cu(II) 3 were practically inactive. Interestingly, theantiproliferative effects of the ligand were only slightly lower thanthose of 1 but significantly higher than those of 4. The sametrend was observed in the rhodamine 123 accumulation. Theaccumulation in MDR1 gene transfected cells was reduced in theorder of 4 o Hp2DAP o 1 in the presence of the compounds.

It is known that the coordination of the metal affects boththe toxicity and the cell penetration of the compounds. Clede36

found a straightforward relationship between the lipophilicity,cytotoxicity and cellular uptake with the length of the sidechain of bio-imaging rhenium triscarbonyl complexes: theincreased lipophilicity increased the penetration, whichincreased the overall toxicity. However, with coordination, theoverall charge distribution and the geometry of the moleculesalso changed. Therefore, metal chelates of Schiff bases mayact as PGP inhibitors.37 The size of the molecules might alsobe crucial38 to their bioavailability. On the other hand, drugresistance is caused by different mechanisms and at the differentstages of the action. One of the reaction mechanisms is mediatedby PGP efflux pump.39

Taking into account all these effects and the calculated MEPand ALIE indicators as measures of the molecule’s reactivity,it is obvious that the specific symmetry, the relatively high reactivesurface and compact structure of complex 1 enhances its biologicalaccessibility. In addition, the most reactive part of the molecule isrelated to the peroxide bridge that may be a source for singlet 1O2

generation. Reactive oxygen species (ROS) play important roles incancer cell functions40 but their role is not yet fully explored.

In Hp2DAP, the charge distribution is more consistent, withoutthe emphasized reaction centres, in comparison with the complexes.In addition, the free ligand is more flexible than its bonded form inthe complexes. Just the opposite is true for the complex of zinc 4wherein the maximum electrostatic potential is located practically atthe metal centre in the inner part of the molecule.

Fig. 8 DSC curves for coordination compounds.

Table 5 Reversal of multidrug resistance of human MDR1 gene trans-fected mouse lymphoma cells in the presence of the Hp2DAP ligand andits complexes

Sample code Conc. (mg cm�3) FSC SSC FL-1 FAR

Verapamil 10 2525 1279 5.86 7.38Hp2DAP 2 2524 1346 6.78 8.531 2 2476 1245 8.49 10.692 2 2463 1248 1.65 2.083 2 2527 1248 1.50 1.894 2 2499 1302 3.98 5.01

where FSC stands for forward scatter count, SSC for side scatter count,FL-1 for fluorescence of the cell population and FAR is the fluorescenceactivity ratio.

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The experimentally determined activities correlate well withthe calculated dipole moment, the polar surface area (PSA), thetotal surface area (TSA), and molecular lipophilicity A log Pvalues (see Table 4). The dipole moment of 1 is by far thelowest (0.24 D) among the compounds. This complex hasthe highest PSA (147 Å2) and TSA (925 Å2) and accordingly,the highest lipophilicity (A log P = 3.70). These parameters showa similar trend in Hp2DAP, which, however, has a significantlyhigher dipole moment (2.23 D), with also relatively high PSAand TSA (120 Å2 and 665 Å2, respectively). Its lipophilicity istherefore lower (A log P = 3.22), but still significantly higherthan those for the corresponding complexes 2–4, which all havevery high, but similar dipole moment (48 D) and small PSAvalues (76 Å2). Their total surface areas are comparable withthat of the ligand (o650 Å2). Among them, the highest TSAbelongs to the zinc complex 4.

Conclusions

A new potentially pentadentate nitrogen donor Schiff baseligand, bis(3-chloropyridazine-6-hydrazon)-2,6-diacetylpyridine,Hp2DAP, was synthesized by the reaction of 3-chloro-6-hydrazino-pyridazine (Hp) and 2,6-diacetylpyridine (DAP). Its coordinationcompounds were obtained in methanolic suspension of the ligandwith Co(II), Ni(II) and Cu(II) acetate solutions, whereas withzinc(II) the reaction was carried out using ZnCl2 and ammonia.The presence of base (acetate ion or ammonia) promoted thedouble deprotonation of the ligand and neutral complexes wereformed. In the case of compounds 2–4, Hp2DAP acted as atetradentate ligand giving mononuclear complexes with square-planar arrangements around the metal centres. By contrast,with cobalt(II) acetate a binuclear octahedral Co(III) complex 1was obtained. The Co(III) centres were additionally linked by aperoxide bridge, which was observed in Co(III) complexes for thefirst time with this type of ligand. A comparison of M–N bondlengths from the CSD showed that the Co(III)–N2 bond wasone of the shortest ones in transition metal complexes with DAPderivatives.

The DFT calculations referred to nitrogen atoms of theligand as suitable centres for electrophilic attacks. In 1, theperoxido bridge was revealed to be the best site for electrophilicreactions along with the nitrogen atoms of the ligand, which isalso true for compounds 2–4. In 4, the highest value of MEP islocated practically on the zinc atom, in accordance with itscoordination preferences, namely, the distorted square-planargeometry is suitable for Ni(II) 2 and Cu(II) 3 but not for Zn(II) 4.

The cytotoxicity of the compounds was low and in the samerange as the reference standard, Verapamil, known also as acalcium channel blocker. The structure–activity relationshipbetween the chemical structures of the compounds and theinhibition of PGP mediated multidrug resistance of cancer cellsis not easily recognized. Complexes 1 and 4 along with theligand Hp2DAP exhibited increased inhibitory effect on ABCtransporter PGP drug efflux pump in the order of 4 o Hp2DAP o 1,whereas complexes 2 and 3 were almost ineffective in the

inhibition of multidrug resistance of cancer cells. The rhodamine123 accumulation in human MDR1 gene transfected tumor cellswas increased in the same order. Complexes 2 and 3 showed noactivity in the flow cytometric studies. This unusual order ofactivity of the compounds can be related to their physico-chemical and structural properties in accordance with thevalues of the calculated MEP and ALIE surfaces.

Acknowledgements

The authors thank to the Ministry of Education, Science andTechnological Development of the Republic of Serbia for financialsupport (continuation of the Projects No. ON172014, OI171039,TR34019) and the Secretariat for Science and TechnologicalDevelopment (Autonomous Province of Vojvodina, Republic ofSerbia). The authors would like to thank MSc Tamas Firkala formeasuring the Raman spectra (Budapest University of Technology,Department of Inorganic and Analytical Chemistry). Moreover, theauthors thank I. Ocsovszky (University of Szeged, Department ofBiochemistry) and A. Csonka (University of Szeged, Departmentof Medical Microbiology and Immunobiology) for the anti-proliferative activity measurements and cytotoxicity tests.Computational part of this study has been performed thanksto the support received from Schrodinger, Inc.

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